Lactobacillus helveticus strain with high ethanol tolerance and application thereof

Abstract

The application discloses a high-ethanol-tolerant Lactobacillus helveticus and application thereof. The Lactobacillus helveticus is named Lactobacillus helveticus FJL4-UV4, and the preservation number is GDMCC NO.62957. The Lactobacillus helveticus is obtained by ultraviolet mutagenesis of a wild Lactobacillus helveticus FJL4 isolated from a Chinese rice liquor fermentation liquor. The Lactobacillus helveticus FJL4-UV4 provided by the application overcomes the defects of the wild Lactobacillus helveticus FJL4 in high-ethanol tolerance, and promotes the generation of ethyl lactate under high-ethanol conditions. The Lactobacillus helveticus FJL4-UV4 is used for solving the problem of low ethyl lactate content in the fermentation liquor in the Chinese rice liquor production process, and the ability of the Lactobacillus helveticus FJL4-UV4 in increasing the ethyl lactate content in the fermentation liquor is significantly improved compared with the wild type.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, and specifically relates to a strain of highly ethanol-tolerant Lactobacillus helveticus and its applications. Background Technology

[0002] Ethyl lactate is an important aroma compound in baijiu (Chinese white liquor). Among the four major esters in baijiu (ethyl acetate, ethyl lactate, ethyl hexanoate, and ethyl butyrate), it is the only ethyl lactate that is miscible with both water and ethanol. In low-alcohol baijiu, it plays a role in eliminating watery tastes and increasing richness, significantly influencing the quality and style of baijiu. Ethyl lactate in baijiu fermentation is produced by the reaction of lactic acid and ethanol, with lactic acid primarily produced by lactic acid bacteria. Ethyl lactate is the most abundant ester in rice-based baijiu (baijiu made primarily from rice and possessing a rice aroma). However, the content of lactic acid and ethyl lactate decreases during the fermentation process of rice-based baijiu due to seasonal and environmental factors. A decrease in ethyl lactate content is a fatal flaw for low-alcohol baijiu.

[0003] Lactic acid bacteria play a crucial role in the formation of flavor compounds and organic acids during the brewing process of alcoholic beverages. However, most lactic acid bacteria have low ethanol tolerance and are unable to exert their effects at high ethanol concentrations. Therefore, selecting high-performance lactic acid bacteria to increase and stabilize ethyl lactate content is of great significance for the production of low-alcohol baijiu. Currently, regarding ethanol-tolerant lactic acid bacteria, patent CN 109468247 B discloses an acid-tolerant lactobacillus isolated from strong-aroma baijiu mash that can tolerate 15% ethanol; patent CN 111378605 B discloses a plant lactobacillus isolated from fruit peel that can tolerate 16% ethanol. However, for rice-based baijiu, existing sequencing results indicate that *Lactobacillus helveticus* is the dominant bacterium in the early and middle stages of fermentation. Utilizing the existing microorganisms in the fermentation system for regulation can reduce the impact on the main flavor.

[0004] To address the problem of low and unstable ethyl lactate content in rice-based baijiu, this patent aims to isolate lactic acid bacteria from baijiu fermentation broth and subject them to mutagenesis to select lactic acid bacteria with high ethanol tolerance, thereby solving the problem of low ethyl lactate content. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain of Lactobacillus helveticus.

[0006] Another object of the present invention is to provide the application of the above-mentioned Lactobacillus helveticus.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A strain of Lactobacillus helveticus is a highly ethanol-tolerant strain obtained by ultraviolet mutagenesis of wild Lactobacillus isolated from rice wine fermentation broth. Its name is Lactobacillushelveticus FJL4-UV4.

[0009] The Lactobacillus helveticus described above has the accession number GDMCC NO.62957 and was deposited on November 11, 2022 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The aforementioned *Lactobacillus helveticus* can grow at a volume concentration of 13.5% ethanol and retains its viability at a volume concentration of 15% ethanol.

[0011] The volume concentration of ethanol in the rice-based liquor fermentation liquid is greater than 13%.

[0012] The application of Lactobacillus helveticus in the fermentation of baijiu (Chinese liquor).

[0013] The aforementioned baijiu refers to rice-based baijiu such as soy sauce aroma baijiu or rice aroma baijiu.

[0014] A method for increasing the ethyl lactate content in rice-based liquor includes the following steps:

[0015] (1) Inoculate Lactobacillus helveticus into MRS broth medium, incubate statically, and centrifuge the bacterial solution in the stable phase to obtain bacterial cells;

[0016] (2) Inoculate Lactobacillus helveticus cells into rice-based liquor fermentation liquid with an alcohol content greater than 13% after ethanol accumulation, and let it stand for culture to increase the ethyl lactate content in rice-based liquor.

[0017] The viable count of *Lactobacillus helveticus* in the stable culture solution described in step (1) is greater than 10. 8 per mL.

[0018] The amount of *Lactobacillus helveticus* inoculated in step (2) should be greater than 10. 7 per mL.

[0019] The present invention has the following advantages and effects compared with the prior art:

[0020] (1) This invention screened and mutated a strain of Lactobacillus helveticus FJL4-UV4, which has better ethanol tolerance than the original wild strain. When applied to rice wine fermentation, it can significantly increase the yield of ethyl lactate.

[0021] (2) The Lactobacillus helveticus FJL4-UV4 obtained in this invention can still grow at a concentration of 13.5% (V / V) ethanol (no Lactobacillus helveticus with stronger ethanol tolerance has been reported so far), indicating that it has excellent ethanol tolerance. Attached Figure Description

[0022] Figure 1 Phylogenetic tree of Lactobacillus helveticus FJL4-UV4.

[0023] Figure 2 This is a photograph of the colony morphology of Lactobacillus helveticus FJL4-UV4.

[0024] Figure 3 This is a photograph of Gram-stained cells (10×100x) of Lactobacillus helveticus FJL4-UV4.

[0025] Figure 4 The OD values ​​of Lactobacillus helveticus FJL4 and FJL4-UV4 at different ethanol concentrations in Example 3 are shown. 600 value.

[0026] Figure 5 In Example 4, Lactobacillus helveticus FJL4 and FJL4-UV4 were used at a ratio of 10... 7 cells / mL and 10 8 The effect of inoculating 1 / mL of rice-based liquor fermentation broth with an ethanol concentration greater than 13% (V / V) on the content of ethyl lactate, lactic acid and reducing sugar during the fermentation process. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0028] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0029] Determination of ethyl lactate:

[0030] The specific gas chromatographic conditions for the determination of ethyl lactate were as follows: column: WondaCap WAX (30m × 0.25mm × 0.25μm); injection port temperature: 250℃; FID detector temperature: 300℃; injection volume: 1μL; split ratio: 10:1; carrier gas: high-purity nitrogen; flow rate: 2.52mL / min. Temperature program: initial temperature 45℃, increased to 55℃ at a rate of 5℃ / min, then increased to 120℃ at a rate of 35℃ / min and held for 4.5min.

[0031] Lactic acid determination:

[0032] The specific liquid chromatography conditions for lactic acid determination were as follows: column: COSMOSIL PBr; mobile phase A: mobile phase B volume ratio: 79:21; mobile phase A was 0.05M phosphate buffer solution with pH=2.5; mobile phase B was methanol; liquid chromatography conditions: isocratic elution for 30 min; flow rate: 0.6 mL / min; injection volume: 10 μL; column temperature: 30℃; detection wavelength: 210 nm.

[0033] Reducing sugars were determined using the DNS method.

[0034] Example 1: Isolation, identification and mutagenesis of Lactobacillus helveticus from Baijiu fermentation broth

[0035] (1) Strain isolation

[0036] The fermented rice-based baijiu (white liquor) from the production base of Jiujiang Winery Co., Ltd. in Nanhai District, Foshan City, Guangdong Province, on the 5th day of fermentation, was shaken at 180 rpm for 10 minutes and diluted 10... 4 10 5 10 6 The culture was spread on MRS broth solid medium supplemented with 2% calcium carbonate and anaerobic incubated at 35°C for 72 hours. Single colonies with calcium dissolution zones were selected. The single colonies were then inoculated into MRS broth medium with Durbin tubes, and non-aerobic strains were selected for later use.

[0037] The MRS broth liquid culture medium formula (per liter) is as follows: 10g casein digest, 10g beef extract, 4g yeast extract, 2g triammonium citrate, 5g sodium acetate, 0.2g MgSO4·7H2O, 0.05g MnSO4·4H2O, 2g dipotassium hydrogen phosphate, 20g glucose, 1.08g Tween 80, with a final pH of 5.7±0.2.

[0038] (2) Strain identification

[0039] The strains isolated in step (1) of this embodiment were subjected to molecular biological identification. The strains were cultured in MRS medium to the logarithmic growth phase, and then DNA was extracted from the test strains using a bacterial genomic DNA extraction kit (Shanghai Sangon Biotech). The 16S rDNA fragments of the obtained microorganisms were amplified using bacterial classification primers (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, 1492R: 5′-GGTTACCTTGTTACGACTT-3′).

[0040] PCR reaction system: 0.5 μL of bacterial genomic DNA, carrying Mg 2+Prepare 2.5 μL of 10×Buffer, 1 μL of dNTP, 0.2 μL of DNA polymerase, and 0.5 μL each of 10 μmol / L forward and reverse primers, then add double-distilled water to a final volume of 25 μL.

[0041] PCR reaction conditions: 94℃ pre-denaturation for 4 min, 94℃ denaturation for 45 s, 55℃ annealing for 45 s, 72℃ extension for 1 min, for a total of 30 cycles; 72℃ repair extension for 10 min, and the reaction was terminated at 4℃.

[0042] The amplified products were subjected to Sanger sequencing, and the obtained sequences were compared with those obtained in NCBI. The results showed that the strain obtained from the above screening was *Lactobacillus helveticus*, and its 16S rDNA was:

[0043]

[0044] (3) Strains mutagenesis and isolation

[0045] Take 20 μL of the bacterial suspension in its growth phase and place it in a 96-well plate. Irradiate the plate with UV light at a distance of 15 cm for 2 min, then place it in the dark for 20 min. Under weak light, add 180 μL of MRS broth containing 13% (v / v) ethanol and shake in the dark for 10 min. Take 100 μL from each well and mix them. Inoculate 1 mL of the mixture into 100 mL of MRS broth containing 14% (v / v) ethanol and incubate at 35°C for 30 days.

[0046] The culture medium from day 30 was diluted and spread onto MRS broth solid medium containing 15% (v / v) ethanol. It was then anaerobically cultured at 35°C for 10 days. Strains with different colony sizes were selected and streaked to obtain single colonies. Single colonies were activated to obtain seed culture, which was then inoculated into MRS broth medium containing 13% (v / v) ethanol and incubated statically at 35°C for 7 days. OD values ​​were then selected. 600 The strain with the highest value was named Lactobacillus helveticus FJL4-UV4 and was deposited on November 11, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNO.62957.

[0047] The obtained strains were sent to a biotechnology company for sequencing and phylogenetic analysis. The results of the sequence phylogenetic analysis are as follows: Figure 1 As shown, this demonstrates that a strain of *Lactobacillus helveticus* FJL4-UV4 was finally obtained. After anaerobic culture at 35°C for 3 days on MRS broth solid medium, strain FJL4-UV4 exhibited a round colony morphology with a slightly convex center, a milky white color, a slightly lighter color at the edges, and a smooth, reflective surface. The colony morphology is as shown in the image. Figure 2 As shown; a single colony is picked and placed on a glass slide. Gram staining is used to observe the cells under a microscope. The cells appear as slender rods, and the Gram stain turns purple, indicating Gram-positive bacteria. Figure 3 As shown.

[0048] Example 2: Genetic stability experiment of Lactobacillus helveticus FJL4-UV4

[0049] Single colonies of *Lactobacillus helveticus* FJL4-UV4 obtained in Example 1 were inoculated into MRS broth medium and incubated statically at 35°C for 48 hours. Then, 2% of the colonies were transferred to fresh MRS broth medium and incubated statically at 35°C for 24 hours to obtain the seed culture. Ethanol was added to the MRS broth medium to achieve an ethanol concentration of 13% (V / V) after inoculation with the 2% seed culture. The culture was then incubated statically at 35°C for 7 days, representing the 0th generation. OD was then measured. 600 The seed culture was transferred at 2% to fresh MRS broth and cultured statically at 35°C for 24 hours. This step was repeated 10 times to obtain Lactobacillus helveticus FJL4-UV4 seed culture after 10 passages. This seed culture was then inoculated into ethanol-containing MRS medium using the same method as the first generation described above, resulting in the 10th generation. OD values ​​were then measured. 600 The results are shown in Table 1. The 10th generation of Lactobacillus helveticus FJL4-UV4 could still grow at a 13% (V / V) ethanol concentration, where wild-type Lactobacillus helveticus FJL4 could not grow, indicating that its mutant-enhanced ethanol tolerance is stably inherited.

[0050] Table 1. OD values ​​of Lactobacillus helveticus at the stationary phase in MRS medium containing 13% (v / v) ethanol for 0 and 10 passages. 600 value

[0051]

[0052] Note: The superscript indicates the number of times the seed liquid has been passaged.

[0053] Example 3: Comparison of ethanol tolerance between mutagenic Lactobacillus helveticus and wild-type Lactobacillus helveticus

[0054] Seed culture was obtained by culturing *Lactobacillus helveticus* FJL4 and its mutant FJL4-UV4, screened in Example 1. Ethanol was added to MRS broth medium to achieve ethanol concentrations of 12%, 12.5%, 13%, 13.5%, and 14% (V / V) after inoculation with the 2% seed culture. Three replicates were established for each ethanol concentration, with a blank control group inoculated with 2% MRS broth medium. The cultures were incubated statically at 35°C for 7 days, and OD was measured. 600 Value minus the OD of the blank culture medium 600 Value is the actual OD 600 Value. Result as follows Figure 4 As shown, Lactobacillus helveticus FJL4-UV4 exhibits significantly improved ethanol tolerance compared to Lactobacillus helveticus FJL4, and the mutant FJL4-UV4 grows better at the same ethanol concentration.

[0055] Example 4: Effects of Mutagenic Lactobacillus helveticus and Wild Lactobacillus helveticus on Ethyl Lactate in Baijiu Fermentation Broth

[0056] 50m from Jiujiang Winery, Guangdong Province 3Five portions of rice-based baijiu fermentation broth with an ethanol concentration greater than 13% (V / V) on day 5 of fermentation were taken from the fermentation tank and divided into five portions (the preparation method of rice-based baijiu fermentation broth can be referred to Example 1 in patent CN113637546A-Rice Aroma Baijiu Production Method). A blank control group (CK) without *Lactobacillus helveticus* was set up. Seed culture of *Lactobacillus helveticus* FJL4 and FJL4-UV4 selected in Example 1 was obtained and transferred at 2% to new MRS broth medium and cultured statically at 35°C for 36 hours until the cell concentration reached approximately 10⁻⁶ cells / mL. 8 The bacterial culture was centrifuged at 8000 rpm to obtain bacterial cell precipitate. The bacterial cell precipitate was then divided into 10... 7 cells / mL and 10 8 Inoculate 1 / mL into the above rice-based liquor fermentation liquid.

[0057] The fermentation broth was incubated at 33℃ for 10 days. During this period, samples were taken to detect the contents of ethyl lactate, lactic acid, and reducing sugars. The results are as follows: Figure 5 As shown, under the same inoculum size, the ethyl lactate content in the culture medium of *Lactobacillus helveticus* FJL4-UV4 was significantly higher than that of *Lactobacillus helveticus* FJL4, especially when the inoculum size of *Lactobacillus helveticus* FJL4-UV4 was 10. 8 The ethyl lactate content increased by 34% when the inoculum size was 10 cells / mL. Simultaneously, the lactic acid production of FJL4-UV4 also increased accordingly. 8 When the reducing sugar content was not completely consumed at 1 / mL, the lactic acid content in the culture medium was the highest. This indicates that adding Lactobacillus helveticus FJL4-UV4 to the fermentation system can improve the conversion rate of reducing sugar to lactic acid in the post-fermentation stage.

[0058] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus helveticus, characterized by: Name is Lactobacillus helveticus FJL4-UV4, with accession number GDMCC NO.62957, was deposited on November 11, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A method for increasing the ethyl lactate content in rice-based liquor, characterized in that... Includes the following steps: (1) Inoculate Lactobacillus helveticus into MRS broth medium, incubate statically, and centrifuge the bacterial solution in the stable phase to obtain bacterial cells; (2) Inoculate Lactobacillus helveticus cells into rice-based liquor fermentation liquid with an alcohol content greater than 13% after ethanol accumulation, and let it stand for culture to increase the ethyl lactate content in rice-based liquor.

3. The method according to claim 2, characterized in that: The viable count of *Lactobacillus helveticus* in the stable culture solution described in step (1) is greater than 10. 8 cells / mL; The amount of *Lactobacillus helveticus* inoculated in step (2) should be greater than 10. 7 per mL.

4. The application of Lactobacillus helveticus as described in claim 1 in the fermentation of Baijiu (Chinese liquor).

5. The application according to claim 4, characterized in that: The liquor mentioned is either soy sauce aroma type or rice aroma type.

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